Notes Bull. Korean Chem. Soc. 2014, Vol. 35, No. 8 2537 http://dx.doi.org/10.5012/bkcs.2014.35.8.2537
Rod and Vesicular Structures of Cyclosophoraose-Based Ionic Self-assembly
Eunae Cho, Daham Jeong, Seung R. Paik,† and Seunho Jung*
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center (BMIC) & Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University, Seoul 143-701, Korea. *E-mail: shjung@konkuk.ac.kr
†Department of Chemical & Biological Engineering, Seoul National University, Seoul 151-742, Korea Received March 15, 2014, Accepted April 5, 2014
Key Words : Quaternary ammonium cyclosophoraose, Self-assembly, Electron microscopy
Cyclosophoraoses (Cys), a class of unbranched cyclic 1,2- β-D-glucans, are produced by many strains of Rhizobium and Agrobacterium.1 They form a variety of large-ring mole- cules that comprise a variable number of glucose residues (17–23) per ring. Conformational analyses have indicated that Cys have flexible backbones and narrow cavities.2 Based on their structure, their ability to form inclusion com- plexes with guest molecules has been studied to investigate their potential as solubility enhancers of poorly soluble guests.3,4 Chemical modification of Cys could also expand its ability for complexation with various drugs.5,6 To the best of our knowledge, here we show for the first time, the self- assembled supramolecular structure formed by Cys, and demonstrate that the secondary structure has the potential for application in development of drug delivery and releasing systems.7,8
Self-assembled materials with well-defined shapes and dimensions are currently attracting considerable interest, and are promising candidates for development of functional molecular devices and nanomaterials.9 Among the various fabrication techniques, ionic self-assembly by coulombic interaction is considered to be a powerful tool for its ease of use, reliability, flexibility, and universality.10 Thus, develop- ment of building blocks for ionic self-assembly is required.
Negatively charged hyaluronan has been investigated for self-assembly with oppositely charged styrylpyridinium or chitosan.11,12 Cationic chitosan has also reported to form nano-assembly with dextran sulfate using ionic interaction.13 In the present study, we introduce a building block based on microbial carbohydrate.
Cys were isolated and purified from Rhizobium trifolii TA-1 as described in the Experimental section. The native Cys was modified with quaternary ammonium (QA), and the synthesized QA–Cys was used as a cationic participant in ionic self-assembly. The modified structures were investi- gated using elemental analysis (EA) and nuclear magnetic resonance (NMR) spectroscopy (Figure 1). Nitrogen con- tents (N%) of QA–Cys were measured to be 4.28 using an elemental analyzer. The degree of substitution (DS) value was determined to be 0.92 by measurement of nitrogen con- tents and calculated according to the following equation:14
DSpract. = %Ncation× 162.15/1401 – (%Ncation× 151.64) In 13C NMR distortionless enhancement polarization trans-
fer (DEPT)-135 spectrum(Figure 1(b)), new peaks were observed and assigned to the methine (C4′), methylenes (C7–9/C7–9′), and quaternary ammonium N-methyl carbons (C10/C10′). In the 1H–13C heteronuclear single quantum coherence (HSQC) spectrum (Figure 1(c)), the C6′ carbons shifted downfield to 72.57 ppm compared to the C6 carbons with a free OH group. In addition, the signal of the H4′
proton was correlated with the corresponding carbon signal at 80.22 ppm. Three cross-peaks at 4.23/76.17, 4.47/67.85, and 3.50/71.00 were detected by the direct correlation between protons and carbons of the –C7,7′H2–, –C8,8′H–, and –C9,9′H2– groups, respectively. The C10,10′/H10,10′
correlations at 3.24 ppm for 1H and at 57.06 ppm for 13C were also assigned. These structural analyses indicated that the novel QA–Cys had been successfully synthesized.
To form the ionic counterpart, Cys was derivatized with carboxymethyl (CM) groups and the structure was confirm- ed, as described in our previous study.6 The native Cys was substituted with CM groups at the OH-4 and OH-6 of the glucose residues with DS of 0.38.
The synthesized cationic and anionic Cys were used as building blocks for ionic self-assembly. Since DS of QA- Cys and CM-Cys are different from each other (DS 0.92 for QA-Cys and DS 0.38 for CM-Cys), the charge imbalance present in the assembled structure is valuable for charged guest molecules. In addition, a carbohydrate backbone can provide a mild amphiphilic character, because of the hydro- phobic or apolar surfaces in its sugar chain, as well as its hydroxyl groups.15,16 Actually, the organic assembled struc- ture is achieved by a variety of forces, including hydrogen bonds, hydrophobic effects, and π-π interactions.17 There- fore, QA-Cys and CM-Cys are considered to make the self- assembled supramolecular structure by electrostatic inter- actions, hydrogen bonding, and hydrophobic effects.
The self-assembled morphologies formed by QA–Cys and CM–Cys were analyzed using scanning electron microscopy (SEM). Figure 2(a) and 2(b) show SEM images of the original QA–Cys and CM–Cys. The surface is flattened and there is no network structure. However, ionic interactions between QA–Cys and CM–Cys, induced to a thickness of 1–
5 µm, showed a branched rod-type morphology (Figure 2(c)); when the molecules were placed in pH 2-buffered solution, the morphology was no longer observed.
In the presence of hexane, self-assembly of the supramole-
2538 Bull. Korean Chem. Soc. 2014, Vol. 35, No. 8 Notes
cular structure was diversified. In the case of 1:20 ratio of hexane to buffer, fiber-type structure was made with 3-5 μm thickness (Figure 2(d)). As increasing the relative concent- ration of hexane, web-like structure of 1-5 μm thickness and bumpy coralliform were observed, respectively (Figure 2(e)
and 2(f)). The organic solvent may have imposed structural distortions or alterations on the supramolecule shown in Figure 2(c).18 These results indicate that novel supramole- cular self-assembled structures were created by coupling of the oppositely charged building blocks (QA–Cys and CM–
Figure 1. Synthetic protocol (a), DEPT-135 spectrum (b), and HSQC spectrum of QA-Cys.
Figure 2. SEM images of QA-Cys (a), CM-Cys (b), and QA-Cys/CM-Cys ionic self-assembly in pH 7 phosphate buffer (c), in 1:20 (d), 1:10 (e), 1:5 (f) hexane : pH 7 phosphate buffer (v/v).
Notes Bull. Korean Chem. Soc. 2014, Vol. 35, No. 8 2539 Cys) and changed by organic solvent. However, the above
supramolecular structures have more surface area and inter- space than unit molecule. Therefore, these self-assembled interwoven structures can provide favourable environment in addition to their respective inclusion complexes to en- capsulate neutral or charged guest molecules.7
For comparison with an anionic counterpart, deoxycholic acid (DCA) was used for ionic self-assembly of QA–Cys.
DCA is a bile acid composed of a steroid skeleton and an acid tail, and well known to possess many exciting bio- logical properties.19 Since QA-Cys could not make self- assembled aggregates with cholesterol, it is proved that the ionic interaction with DCA is critical step forming the supra- molecule.
The self-assembly formed by QA–Cys and DCA in aque- ous solution was revealed using transmission electron micro- scopy (TEM). Figure 3(a) and 3(b) show clear spherical micro-aggregates with vesicular shells, the morphology of which contrasts strongly with the morphology of QA–Cys/
CM–Cys. The diameters ranged from approximately 100–
200 nm in the TEM image; results of dynamic light scatter- ing (DLS) showed diameters from 150–300 nm (Figure 3(c)). The discrepancy may be accounted for by noting that TEM and DLS show solid and swollen vesicles, respec- tively.20 Anthraquinone modified β-cyclodextrin and DCA- modified chitosan have also been used for carrying drugs and DNA with the self assembled spherical structures of 200 nm and 130-300 nm in diameter, respectively.7,21 Although the synthetic amphiphilic carbohydrates have been reported as the building block for self-assembled vesicle,7,21,22 the vesicle based on electrostatic interactions has not been eluci- dated. The present self-assembled vesicular structure repre- sents a novel phenomenon based on the ionic interactions between hydrophobic anion and cationic carbohydrate.
Furthermore, these self-assembled vesicles can be used for
biomimetic models, drugs or gene carriers, nanoreactors.23-25 In conclusion, Cys isolated from Rhizobium trifolii TA-1 were modified with quaternary ammonium groups and carboxymethyl moieties through chemical derivatization, respectively. Their modified structures were characterized with NMR, EA, and MALDI-TOF MS. The QA–Cys and CM–Cys were used as novel oppositely charged carbohyd- rate building blocks for self-assembled structures. Electro- static attraction between the cationic and anionic portions produced ionic self-assembly behavior, and the resulting supramolecular structure and morphology were analyzed with electron microscopy (SEM and TEM). Furthermore, the self-assembled morphology could be controlled by changing the reaction conditions (pH, solvent, and addition of another anion). The present microbial carbohydrate-based supramolecule can be applied to tunable self-assembling materials for eco-friendly electronic devices or drug/gene delivery systems with its dual functionality as the macro- cyclic host and supramolecular architecture. Further studies of applications using these supramolecules are in progress.
Experimental
Purification of Cys. The isolation and purification of Cys from Rhizobium trifolii TA-1 were carried out as previously described.26 Culture supernatants were concentrated fivefold by rotary evaporation, and the concentrated sample was precipitated by adding 3 volumes of ethanol. After centrifu- gation, the supernatant was concentrated by rotary evapo- ration and the product was collected by adding 7 volumes of ethanol. After decanting the supernatant, the precipitates were applied to Bio-Gel P-6 column. The fractions were assayed for carbohydrates using the phenol-sulfuric acid method. The fractions that contained Cys were pooled, concentrated, and desalted using Bio-Gel P-4 column. The desalted Cys were confirmed by NMR spectroscopy and MALDI-TOF MS.
Synthesis of QA–Cys. Cys (1 g) was dissolved in 7%
NaOH aqueous solution for 30 min, and 2,3-epoxypropyl- trimethylammonium chloride (3.1 g) was added. The mix- ture was reacted for 5 h at 65 °C, neutralized with HCl and evaporated to a viscous residue. After desalting on a Bio-gel P-2 column, the product was lyophilized, and the final struc- ture was elucidated with EA (ThermoFinnigan, Flash2000) and NMR spectroscopy.
Synthesis of CM–Cys. CM–Cys was prepared as describ- ed in a previous report.6 Cys (500 mg) and NaOH (2.8 g) were mixed in distilled water (7.4 mL). Monochloroacetic acid solution (20%) was added. After reacting for 4 h at 50
°C, the mixture was neutralized with 6 M HCl. The mixture was precipitated with 5 vol MeOH and left overnight at 4
°C. The precipitate was dissolved in water and desalted on a Sephadex G-10 column. The product was confirmed using MALDI-TOF MS (Voyager-DETM STR Biospectrometry Workstation) and NMR spectroscopy.
Nuclear Magnetic Resonance (NMR) Spectroscopy. For the NMR spectroscopic analysis, a Bruker Avance 500 Figure 3. TEM image (a), enlarged image (b) (scale bars = 200
nm), and DLS profile of QA-Cys/DCA ionic self-assembly in water.
2540 Bull. Korean Chem. Soc. 2014, Vol. 35, No. 8 Notes spectrometer was used to record the 1H–NMR, 1C-NMR,
DEPT and HSQC spectra. The HSQC spectrum was mea- sured with a spectral width of 3401 Hz in both dimensions and 256/2048 complex data points in t1 and t2, respectively.
NMR analyses were performed in D2O at room temperature.
Scanning Electron Microscopy (SEM). QA–Cys (3 mM) and CM–Cys (3 mM) were mixed in 100 μL of 20 mM phosphate buffer (pH 7). Hexane was added to a QA–Cys/
CM–Cys mixture in phosphate buffer (pH 7), and a turbid solution was obtained by vortexing for 5 min. After centrifu- gation, the precipitate was lyophilized. The lyophilized samples were mounted onto stubs using double-sided ad- hesive tape and then made electrically conductive by coating with a thin layer of gold. The surface morphologies of the materials were examined under a scanning electron micro- scope (Jeol, JSM 6380, Tokyo, Japan).
Transmission Electron Microscopy (TEM). QA–Cys (3 mM) and DCA (3 mM; Sigma Aldrich) were mixed in 800 μL of 20 mM phosphate buffer (pH 7). After sonication for 30 s, the aqueous suspension (10 µL) containing the supra- molecular aggregates formed by the QA–Cys/DCA mixture was adsorbed onto a carbon-coated copper grid (300-mesh) and air-dried for 1 min. For clear negative staining, the supernatant of 2% uranyl acetate following centrifugation at 13,200 rpm for 2 min was used. The aggregates were ex- amined using a transmission electron microscope (JEOL, JEM 1010, Tokyo, Japan).
Dynamic Light Scattering (DLS). DLS measurements were carried out with a Wyatt Technology DynaPro Plate Reader at constant room temperature.
Acknowledgments. This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A1A2012568 and NRF- 2011-619-E0002) and supported by the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-0006686). SDG.
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